Tubulysin conjugates

ABSTRACT

The invention relates to novel tubulysin conjugates (e.g. of tubulysin A) and the use thereof in the treatment of cancer diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application under 35 U.S.C.§371 of International Application No. PCT/EP03/07415, filed Jul. 9, 2003which claims the benefit of German Application No. 103 05 531.2, filedFeb. 11, 2003 and German Application No 102 30 875.6, filed Jul. 9,2002, each which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to novel tubulysin conjugates and the usethereof in the treatment of cancer diseases.

The tubulysins were for the first time isolated by the group of Höfleand Reichenbach (GBF Braunschweig) from a culture broth of strains ofthe Myxobacteria Archangium gephyra (F. Sasse et al. J. Antibiot. 2000,53, 879-885; WO9813375; DE 10008089). These compounds show an extremelyhigh cytotoxic activity against mammalian cell lines with IC50 values inthe picomolar range and therefore, are of high interest as potentialtherapeutics against cancer.

The extremely high cytotoxicity of some tubulysins also exhibitsdisadvantages: a high general toxicity as well as a low selectivityagainst normal cells.

The object of the present invention is to lower the toxicity of thetubulysins and to enhance their selectivity.

SUMMARY OF THE INVENTION

The present invention provides tubulysin conjugates of the generalformula U—V—W, wherein U represents the Formula (I),

wherein

-   A is a optionally substituted five or six membered heteroaromatic    system;-   X is an oxygen atom, a sulfur atom, a group of the formula NR13 or    CR14R15;-   Y is an oxygen atom, a sulfur atom or a group of the formula NR16    and the terms R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,    R¹³, R¹⁴, R¹⁵ and R¹⁶ independently from each other a hydrogen atom,    a alkyl-, alkenyl-, alkynyl-, heteroalkyl-, aryl-, arylalkyl-,    heteroaryl-, heteroarylalkyl-, cycloalkyl-, heterocycloalkyl-,    alkylcycloalkyl- or heteroalkylcycloalkyl-group or two of the groups    together are part of a cycloalkyl- or heterocycloalkyl ring system,    V is a linker and W is a polymer or a biomolecule.

In the present invention it was found that polymer conjugates andbioconjugates, resp., of special tubulysins exhibit a higher selectivityby a given cyctotoxicity as well as lower toxicity as the unconjugatedcompounds. The result is that predominantly cancer cells are targeted inthe human and the animal body and healthy tissue is not affected.

DETAILED DESCRIPTION OF THE INVENTION

The term alkyl refers to a saturated straight or branched chain alkylgroup, containing from one to twenty carbon atoms, preferably from oneto twelve carbon atoms, especially preferred from one to six carbonatoms, for example methyl, ethyl, propyl, iso-propyl, iso-butyl,tert.-butyl, n-hexyl, 2,2-dimethylbutyl or n-octyl groups.

The terms alkenyl and alkinyl refer to at least partly unsaturatedstraight or branched chain alkyl groups, containing from two to twentycarbon atoms, preferably from two to twelve carbon atoms, especiallypreferred from two to six carbon atoms, for example ethenyl, propenyl,iso-propenyl, isoprenyl or hexa-2-enyl. Preferably exhibit alkenylgroups one or two (especially preferred one) double bonds resp. alkynylgroups one or two (especially preferred one) triple bonds.

The terms alkyl, alkenyl and alkynyl moreover refer to groups, whereinone or more hydrogen atoms are replaced by halogen atoms (preferablyfluorine or chlorine) such as, for example the 2,2,2-trichloroethyl orthe trifluoromethyl groups

The term heteroalkyl refers to an alkyl, a alkenyl, or a alkynyl groupas defined herein where one or more (preferably 1, 2 or 3) carbon atomsare replaced by an oxygen, nitrogen, phosphorous, boron, selenium,silicium or sulphur atom (preferably oxygen, sulfur or nitrogen). Theterm heteroalkyl furthermore refers to a caboxylic acid or a groupderived from a carboxylic acid such as for example acyl (alkyl-CO—),acylalky, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide oralkoxycarbonyloxy.

Examples for heteroalky groups are groups of the formulas

-   R^(a)—O—Y^(a)—, R^(a)—S—Y^(a)—, R^(a)—N(R^(b))—Y^(a)—,    R^(a)—CO—Y^(a)—, R^(a)—O—CO—Y^(a)—, R^(a)—CO—O—Y^(a),    R^(a)—CO—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CO—Y^(a)—,    R^(a)—O—CO—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CO—O—Y^(a)—, R^(a—N(R)    ^(b))—CO—N(R^(c))—Y^(a)—, R^(a)—O—CO—O—Y^(a)—,    R^(a)—N(R^(b))—C(═NR^(d))—N(R^(c))—Y^(a)—, R^(a)—CS—Y^(a)—,    R^(a)—O—CS—Y^(a)—, R^(a)—CS—O—Y^(a)—, R^(a)—CS—N(R^(b))—Y^(a)—,    R^(a)—N(R^(b))—CS—Y^(a)—, R^(a)—O—CS—N(R^(b))—Y^(a)—,    R^(a)—N(R^(b))—CS—O—Y^(a)—, R^(a)—N(R^(b))—CS—N(R^(c))—Y^(a)—,    R^(a)O—CS—O—Y^(a)—, R^(a)—S—CO—Y^(a)—, R^(a)—CO—S—Y^(a)—,    R^(a)—S—CO—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CO—S—Y^(a)—,    R^(a)—S—CO—O—Y^(a)—, R^(a)—O—CO—S—Y^(a)—, R^(a)—S—CO—S—Y^(a)—,    R^(a)—S—CS—Y^(a)—, R^(a)—CS—S—Y^(a)—, R^(a)—S—CS—N(R^(b))—Y^(a),    R^(a)—N(R^(b))—CS—S—Y^(a)—, R_(a)—S—CS—S—Y^(a), R^(a)—O—CS—S—Y^(a)—,    wherein R^(a) is a hydrogen atom, a C₁-C₆-alkyl-, a C₂-C₆-alkenyl or    a C₂-C₆-alkynyl group; R^(b) is a hydrogen atom, a C₁-C₆-alkyl-, a    C₂-C₆-alkenyl or a C₂-C₆-alkynyl group; R^(c) is a hydrogen atom, a    C₁-C₆-alkyl-, a C₂-C₆-alkenyl or a C₂-C₆-alkynyl group; R^(d) is a    hydrogen atom, a C₁-C₆-alkyl-, a C₂-C₆-alkenyl or a C₂-C₆-alkynyl    group and Y^(a) is a direct bond, a C₁-C₆-alkylene-, a    C₂-C₆-alkenylene or a C₂-C₆-alkynylene group, wherein each    heteroalkyl group contains at least one carbon atom and one or more    hydrogen atoms can be replaced by fluoro or chloro atoms. Concrete    examples for heteroalky groups are methoxy, trifluoromethoxy,    ethoxy, n-propyloxy, iso-propyloxy, tert.-butyloxy, methoxymethyl,    ethoxymethyl, methoxyethyl, methylamino, ethylamino, dimethylamino,    diethylamino, iso-propylethylamino, methylaminomethyl,    ethylaminomethyl, di-iso-propylaminoethyl, enolether,    dimethylaminomethyl, dimethylaminoethyl, acetyl, propionyl,    butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl,    N-ethyl-N-methylcarbamoyl or N-methylcarbamoyl.

Further examples for heteroalky groups are nitrile, isonitrile, cyanate,thiocyanate, isocyanate, isothiocyanate and alkylnitrile groups.

The term cycloalky refers to a saturated or partially unsaturated (e.g.cycloalkenyl) cyclic group which comprises of one or several rings(perferably 1 or 2) which contain in total three to fourteen ring carbonatoms, preferably three to ten (especially 3, 4, 5, 6 or 7) ring carbonatoms. The term cycloalkyl refers also to such groups in which one ormore hydrogen atoms are replaced by fluoro, chloro, bromo or iodo atomsor OH, ═O, SH, ═S, NH₂, =NH, or NO₂ groups, e.g. cyclic ketones such ase.g. cyclohexanone, 2-cyclohexenone or cyclopentanone. Additionalconcrete examples for cycloalkyl groups are the cyclopropyl, cyclobutyl,cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclopentenyl,cycloheadienyl, decalinyl, cubanyl, bicyclo[4.3.0]nonyl, tetralin,cyclopentylcyclohexyl, fluorocyclohexyl or the cyclohex-2-enyl group.

The term heterocycloalkyl refers to a cycloalkyl group as difined abovein which one or more (preferably 1, 2 or 3) ring carbon atoms arereplaced by an oxygen, nitrogen, silicon, selenium, phosphorous orsulfur atom (preferably oxygen, sulfur or nitrogen). Preferably theheterocycloalky group contains 1 or 2 rings with 3 to 10 (especially 3,4, 5, 6 or 7) ring atoms. The term heterocycloalkyl refers also to suchgroups in which one or more hydrogen atoms are replaced by fluoro,chloro, bromo or iodo atoms or OH, ═O, SH, ═S, NH₂, =NH, or NO₂ groups.Examples thereof are the piperidyl, morpholinyl, urotropinyl,pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl,oxacyclopropyl, azacyclopropyl or 2-pyrazolinyl group as well aslactams, lactones, cyclic imides and cyclic anhydrides.

The term alkylcycloalkyl refers to groups which according to thedefinitions given above contain either cycloalkyl and alky, alkenyl oralkynyl groups, e.g. alkylcycloalkyl, alkylcycloalkenyl,alkenylcycloalkyl and alkynylcycloalkyl groups. Preferably aalkylcyckloalkyl group contains a cycloalkyl group which has one or tworing systems which in total comprises 3 to 10 (especially 3, 4, 5, 6 or7) carbon atoms and one or two alkyl, alkenyl or alkynyl groups with 1or 3 to 6 carbon atoms.

The term heteroalkylcycloalkyl refers to alkylcycloalkyl groups in whichaccording to the definitions given above one or more (preferably 1, 2 or3) carbon atoms are replaced by an oxygen, nitrogen, silicon, selenium,phosphorous or sulfur atom (preferably oxygen, sulfur or nitrogen).Preferably the heteroalkylcycloalky group contains 1 or 2 ring systemswith 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms and one or twoalkyl, alkenyl, alkynyl or heteroalkyl groups with 1 or 2 to 6 carbonatoms. Examples of such groups are alkylheterocycloalkyl,alkylheterocycloalkenl, alkenylheterocycloalkyl, akynylheterocycloalkyl,heteroalkylcycloalkyl, heteroalkylheterocycloalkyl andheteroalkylheteocycloalkenyl in which the cyclic groups are saturated orsingle, double or triple unsaturated.

The term aryl and Ar, resp., refer to a aromatic group which has one ormore rings which in total contain 6 to 14 ring carbon atoms, preferably6 to 10 (especially 6) ring carbon atoms. The term aryl (resp. Ar) referfurther to such groups in which one or more hydrogen atoms are replacedby fluoro, chloro, bromo or iodo atoms or OH, SH, NH₂ or NO₂ groups.Examples are the phenyl, naphthyl, 2-fluorophenyl, anilinyl,3-nitrophenyl or 4-hydroxyphenyl group.

The term heteroaryl refers to a aromatic group which has one or morerings which in total comprise 5 to 14 ring atoms, preferably 5 to 10(especially 5 or 6) ring atoms and one or more (preferably 1, 2, 3 or 4)oxygen, nitrogen, phosphorous or sulfur ring atoms (preferably O, S orN). The term heteroaryl refers further on such groups in which one ormore hydrogen atoms are replaced by fluoro, chloro, bromo or iodo atomsor OH, SH, NH₂ or NO₂ groups. Examples are 4-pyridyl, 2-imidazolyl,3-phenylpyrrolyl, thiazolyl, oxazolyl, triazolyl, tetrazolyl,isoxazolyl, indazolyl, indolyl, benzimidazolyl, pyridazinyl, chinolinyl,purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3′-bifuryl, 3-pyrazolyl andisochinolinyl groups.

The term aralkyl refers to groups which according to the abovedefinitions contain either aryl and alkyl, alkenyl, alkynyl and/orcycloalkyl groups such as e.g. arylalkyl, arylalkenyl, arylalkynyl,arylcycloalkyl, arylcycloalkenyl, alkylarylcycloalkyl andalyklarylcycloalkenyl groups. Concrete examples for aralkyls aretoluene, xylene, mesitylene, styrole, benzylchloride, o-fluorotoluene,1H-indene, tetraline, dihydronaphthaiine, indanone, phenylcyclopentyl,cumol, cyclohexylphenyl, fluorene and indan. Preferably a aralkyl groupcontains one or two ring systems (1 or 2 rings) with in total 6 to 10ring carbon atoms and one or two alkyl, alkenyl and/or alkynyl groupswith 1 or 2 to 6 carbon atoms and/or a cycloalkyl group with 5 or 6 ringcarbon atoms.

The term heteroaralkyl refers to a aralkyl group as defined above inwhich one or more (preferably 1, 2, 3 or 4) carbon atoms are replaced byoxygen, nitrogen, silicon, selenium, phosphorous, boron or sulfur atom(preferably oxygen, sulfur or nitrogen), i.e. to groups which accordingto the definitions above contain either aryl or heteroaryl resp., andalkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/orheterocycloalkyl groups. Preferably a heteroaralkyl group contains oneor two aromatic ring systems (1 or 2 rings) with in total 5 or 6 to 10ring carbon atoms and one or two alkyl, alkenyl and/or alkynyl groupswith 1 or 2 to 6 carbon atoms and/or a cycloalkyl group with 5 or 6 ringcarbon atoms whereby 1, 2, 3 or 4 of these carbon atoms are replaced byoxygen, sulfur or nitrogen atoms.

Examples are arylheteroalkyl, arylheterocycloalkyl,arylheterocycloalkenyl, arylalkylheterocycloalkyl,arylalkenylheterocycloalkyl, arylalkynylheterocycloalkyl,arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl,heteoarylalkynyl, heteroarylheteroalkyl, heteroarylcycloalkyl,heteroarylcycloalkenyl, heteroarylheterocycloalkyl,heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl,heteroarylalkylheterocycloalkenyl, heteroarylheteroalkylcycloalkyl,heteroarylheteroalkylcycloalkenyl andheteroarylheteroalkylheterocycloalkyl groups, whereby the cyclic groupsare saturated or single, double or triple unsaturated. Concrete examplesare the tetrahydroisochinolinyl, benzoyl, 2- or 3-ethylindolyl,4-methylpyridino, 2-, 3- or 4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or4-carboxyphenylalkyl group.

The terms cycloalkyl, heterocycloalkyl, alkylcycloalkyl,heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl referalso to groups in which one or more hydrogen atoms of such groups arereplaced by fluoro, chloro, bromo or iodo atoms or OH, ═O, SH, ═S, NH₂,=NH, or NO₂ groups.

The term “optionally substituted” refer to groups in which one or morehydrogen atoms are replaced by fluoro, chloro, bromo or iodo atoms orOH, ═O, SH, ═S, NH₂, =NH, or NO₂ groups. This term also refer to groupswhich are exclusively or additionally substituted with unsubstitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₁₀cycloalkyl, C₂-C₉ heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₉ heteroaryl,C₇-C₁₂ aralkyl or C₂-C₁₁ heteroalkyl groups.

The term linker refers to a group which is used to connect compounds ofthe formula (I) with a polymer or a biomolecule. A linker can be adirect bond, a alkylene, alkenylen, alkynylene heteroalkylene, arylene,heteroarylene, cycloalkylene, alkylcycloalkylene,heteroalkylcycloalkylene, heterocycloalkylene, aralkylene or aheteroaralkylene. Preferred are linkers which are stable towards bloodplasma (especially stable towards hydrolysis), metabolically cleavableand after the cleavage non-toxic.

Examples for linkers are described in P. Seneci, Solid-Phase Synthesisand Combinatorial Technologies, John Wiley & Sons, New York 2000; D.Obrecht and J. M. Villalgordo, Solid-Supported Combinatorial andParallel Synthesis of Small-Molecular-Weight Compound Libraries,Tetrahedron Organic Chemistry Series Volume 17, Elsevier Science Ltd.,Oxford, 1998 as well as in F. Z. Dörwald, Organic Synthesis on SolidPhase, Wiley-VCH, Weinheim, 2000, those examples are included underreference. As examples are named here the trityl linker, the Wanglinker, the SASRIN™-linker, the Rink acid linker, the benzhydrylalkohollinker, the HMBA-linker as well as polymeric benzylhalogenid (linker ofthe Merrifield resin) and the PAM-linker.

As polymers especially synthetic polymers come into question such ase.g. polyethylenglycol (MW=200, 300, 400, 30,000, 35,000, 40,000;especially MW=25,000-100,000 Da, perferably 25,000-50,000 Da),polyethylenglycol dendrimers, polyacrylic acid, hydroxyethyl starch(HES), polylactic-glycolid, poly-D,L-lactic acid-p-dioxanonepolyethyleneglycol block copolymer (PLA-DX-PEG), poly(ortho) ester, polyglutamate,polyaspartate, polymer from α-β-unsaturated monomers: (meth)acrylicacid, crotonic acid, maleic acid, maleic anhydride, fumaric acid,itaconic acid/anhydride, etc. Comonomers including: vinyl ether, vinylester, vinyl amide, olefins, diallyl-dialkyl-ammonium-halogenes,prefered vinyl ether, poly-(diethylene glycol adipat), poly ethyleneimine, poly glycolide, poly urea, poly limonene (=polylimo), poly(2-methyl-1,3-propylene adipat), grafted polymers, graft (block-)polymers with othe polymers.

As biomolecules e.g. glycoproteins, lipoproteins, lectins, hormones e.g.somatostatin and somatostatin analogues, resp., synthetic hormonanalogues, albumine, liposomes, DNA, dextrane, biotin, streptavidine,avidine, cells or antibodies come into question. Preferably thebiomolecule is an antibody, more preferred a monoclonal antibody (e.g.herceptine).

Further examples for linkers, polymers and biomolecules are described inG. T. Hermanson, Biokonjugate Techniques, Academic Press, San Diego,1996, whose examples therein are included under reference.

It should be appreciated that compounds of the formula (I) because oftheir substitution may contain one or more chirality centers. Therefore,the present invention relates either all possible pure enantiomers andall possible pure diastereoisomers as well as their mixtures in everymixing ratio.

Preferred X is a CH₂ group.

Moreover preferred Y is an oxygen atom.

Further preferred A has the following structure:

Furthermore preferred R¹ and R³ are together part of a cycloalkyl ring;more preferred R¹ and R³ together are of the formula —(CH₂)₄—.

Further preferred R² is a C₁-C₄ alkyl group, more preferred a methylgroup.

Furthermore preferred R⁴, R⁵, R⁶ and R¹⁰ are hydrogen atoms.

Moreover preferred R⁷ is a alkyl group; more preferred a group of theformula —CH(CH₃)CH₂CH₃.

Furthermore preferred R⁸ is a hydrogen atom, a alkyl, alkenyl or aheteroalkyl group; more preferred a group of the formula —CH₂C(═O)R¹⁷,whereby R¹⁷ is a C₁-C₆ alkyl or a C₂-C₆ alkenyl group.

Moreover preferred R⁹ is a alkyl group; more preferred a group of theformula —CH(CH₃)₂.

Further preferred R¹¹ is a hydrogen group or a acetyl group.

Further preferred R¹² is a group of the formula NHR¹⁸ whereby R¹⁸ is aheteroalkyl group.

More preferred R¹⁸ has the following structures:

wherein R¹⁹ and R²⁰ independentely from each other are hydrogen atoms,OH, NH₂, alkoxy, alkenyloxy, alkynyloxy, heteroalkyl, aryloxy,heteroaryloxy, cycloalkyloxy, alkylcycloalkyloxy, heteroalkylcycloalkyl,heterocycloalkyloxy, aralkyloxy or heteroaralkyl groups; more preferredR¹⁹ and R²⁰ are hydroxy groups. In case the phenyl ring of both of thesestructures can be substituted (e.g. by a NO₂ group).

More preferred the compound of formula (I) is Tubulysin A.

Further preferred is the linker connected to compounds of the formula(I) via the residues R⁸, R¹¹, R¹⁹ or R²⁰; more preferred via theresidues R¹⁹ or R²⁰.

Further preferred the polymer is a polyethyleneglycol PEG (especially aPEG with a molecular weight of more than 30 kDa to 100 kDa, preferred ofmax. 50 kDa), which especially is bound to Tubulysin A via R²⁰ (in casewith a linker V).

More preferred the conjugates (U—V—W) in the present invention comprisethe following formula wherein the stereochemistry corresponds to the oneof the natural Tubulysin A:

Preferred V is an oxygen atom, a NH group or a group of the formula—O—(CR^(a)R^(b))_(n)—O— (whereby R^(a) and R^(b) are independently fromeach other C₁-C₆ alkyl groups or together part of cycloalkyl group and nis 1 or 2), —NH—R^(c)—NH—CO—CH₂—O—, —O—R^(c)—O—CO—CH₂—O— or a group ofthe formula —O—R^(c)—O— (whereby R^(c) is a alkylene, arylene or acycloalkylene group).

Pharmacologically acceptable salts, solvates, hydrates or formulationsof the herein described conjugates are also part of the presentinvention. Examples for pharmacologically acceptable salts of compoundsof the formula (I) are salts of physiologically acceptable mineralicacids such as hydrochloric acid, sulfuruc acid and phosphoric acid orsalts of organic acids such as methansulfonic acid, p-toluenesulfonicacid, lactic acid, acetic acid, formic acid, trifluoroacetic acid,citric acid, succinic acid, fumaric acid, maleinic acid and salicylicacid. Compounds of the formula (I) can be solvated, especially hydrated.The hydratisation can occur e.g. during the preparation process or asconsequence of the hygroscopic nature of the initially water freecompounds of the formula (I).

The pharmaceutical compositions in the present invention contain atleast one compound of the formula (I) as active ingredient andoptionally carriers and/or adjuvants.

The therapeutic use of compounds of the formula (I), theirpharmacologically acceptable salts and solvates, resp. and hydrates aswell as formulations and pharmaceutical compositions is also part of thepresent invention.

The use of these active ingredients for the manufacturing of drugs isalso part of the present invention. Furthermore, the present compoundsare of great interest for the prevention and/or treatment of fungalinfections (i.e. as antifungal agents), rheumatoid arthritis,inflammatory diseases, immunologically caused diseases (e.g. diabetestype 1), autoimmuno diseases as well as tumor diseases. In generalcompounds of the formula (I) will be administered by using the known andacceptable modes known in the art, either alone or in combination withany other therapeutic agent. Such therapeutically useful agents can beadministered preferably parenteral, e.g. as injectable solution. For theproduction of liquid solutions one may use excipients as are e.g. water,alcohols or aqueous saline. The pharmaceutically useful agents may alsocontain additives for conservation, stabilisation, salts to change theosmotic pressure, buffers and antioxidants.

Combinations with other therapeutic agents may contain further activeingredients which are usually employed in the therapy of tumor diseases.

EXAMPLES

To a solution of 0.056 mmol Tubulysin A and 0.125 mmol PEG (6 kDa, 10kDa, 20 kDa, 35 kDa and 40 kDa, resp.) in a mixture of 3 ml acetonitrileand 1 ml of DMF was added at 0° C. 0.1 mmol 2-chloro-1-methylpyridiniumiodide (Mukaiyama reagent) and 0.2 mmol 4-dimethyl aminopyridine (DMAP).The reaction mixture was stirred for 2 h at 0° C. and another 60 h atroom temperature. Then the mixture was evaporated, dissolved in 15 ml ofdichloromethane and washed with each 5 ml water, NaHCO₃ (aq), water andsaturated sodium chloride solution. The organic phase was dried overNa₂SO₄ and the solvent was removed under reduced pressure. The residuewas dissolved in 8 ml dichloromethane and under stirring dry ether wasadded until the solution became turbid and was allowed to stand for onehour. The solid was collected by filtration and washed with ether. Thedesired products were obtained as white powders.

The diamines of the polyethyleneglycols (6 kDa, 10 kDa, 20 kDa, 35 kDabzw. 40 kDa) as well as their conjunction with Tubulysin A were preparedin accordance to the procedure described in R. B. Greenwald et al.Bioorg. Med. Chem. 1998, 6, 551-562.

The PEG dicarbonic acids (6 kDa, 10 kDa, 20 kDa, 35 kDa bzw. 40 kDa) aswell as their conjugation with Tubulysin A were prepared in accordanceto the procedure described in R. B. Greenwald et al. J. Med, Chem. 1996,39, 424-431.

1. A compound in the general formula U—V—W, wherein U refers to the Formula (I),

wherein A has the following structure

X is CH₂; Y is an oxygen atom; R¹ and R³ together are of the formula —(CH₂)₄—; R² is a C₁-C₄ alkyl group; R⁴, R⁵, R⁶, and R¹⁹ are hydrogen atoms; R⁷ is an alkyl group; R⁸ is a hydrogen atom, an alkyl, alkenyl, or a heteroalkyl group R⁹ is an alkyl group; R¹¹ is an acetyl group; R¹² is a group of formula NHR¹⁸; R¹⁸ has the following structures:

wherein R¹⁹ is H or OH and R²⁰ is —V—W or wherein R¹⁹ is —V—W and R²⁰ is OH, NH₂, or a heteroalkyl group; V is an oxygen atom, a NH group, or a heteroalkylene group wherein the heteroatoms are selected from O, S, and N; and W is polyethylene glycol (PEG) or a cyclodextrin comprising polyethylene glycol (PEG).
 2. A method for treating a patient suffering from breast cancer, cervical cancer, ovarian cancer, colorectal cancer or non-small cell lung cancer, comprising administering to the patient one or more compounds of claim
 1. 3. A compound of claim 1 having the following formula:

wherein V is an oxygen atom; a NH group; a group of the formula —O—(CR^(a)R^(b))_(n)—O— where R^(a) and R^(b) are independently C₁-C₆alkyl groups or together part of cycloalkyl group and n is 1 or 2; —NH—R^(c)—NH—CO—CH₂—O—; —O—R^(c)—O—CH₂—O—; or a group of the formula —O—R^(c)—O— where R^(c) is alkylene, arylene or a cycloalkylene group.
 4. The compound of claim 3 wherein V is oxygen.
 5. The method of claim 2 wherein a compound having the following formula is administered:

wherein V is an oxygen atom; a NH group; a group of the formula —O—(CR^(a)R^(b))_(n)—O— where R^(a) and R^(b) are independently C₁-C₆alkyl groups or together part of cycloalkyl group and n is 1 or 2; —NH—R^(c)—NH—CO—CH₂—O—; —O—R^(c)—O—CH₂—O—; or a group of the formula —O—R^(c)—O— where R^(c) is alkylene, arylene or a cycloalkylene group.
 6. The method of claim 5 wherein V is oxygen.
 7. The method of claim 5 wherein V is a NH group.
 8. The method of claim 5 wherein V is a group of the formula —O—(CR^(a)R^(b))_(n)—O—.
 9. The method of claim 5 wherein V is an oxygen atom; a NH group; a group of the formula —O—(CR^(a)R^(b))_(n)—O— where R^(a) and R^(b) are independently C₁-C₆alkyl groups or together part of cycloalkyl group and n is 1 or 2; —NH—R^(c)—NH—CO—CH₂—O—; —O—R^(c)—O—CH₂—O—; or a group of the formula —O—R^(c)—O— where R^(c) is alkylene, arylene or a cycloalkylene group.
 10. The method of claim 5, wherein the polyethylene glycol has a molecular weight of 30 kDa.
 11. The method of claim 5, wherein the polyethylene glycol has a molecular weight of 35 kDa.
 12. A compound, according to claim 1, wherein R² is a methyl group.
 13. A compound, according to claim 1, wherein R⁷ is a group of formula —CH(CH₃)CH₂CH₃.
 14. A compound, according to claim 1, wherein R⁸ is a hydrogen atom or a group of formula —CH₂OC(═O)R¹⁷, wherein R¹⁷ is a C₁-C₆ alkyl or a C₂-C₆ alkenyl group.
 15. A compound, according to claim 1, wherein R⁹ is a group of formula —CH(CH₃)₂.
 16. A compound, according to claim 1, wherein V is an oxygen atom, a NH group, or a group of the formula —O—(CR^(a)R^(b))_(n)—O—, whereby R^(a) and R^(b) independently from each other are C₁-C₆ alkyl groups, or, together, are part of a cycloalkyl group and n is 1 or 2; —NH—R^(c)—NH—CO—CH₂—O—, —O—R^(c)—O—CO—CH2-O—, or a group of formula —O—R^(c)—O—, whereby R^(c) is an alkylene, arylene, or a cycloalkylene group.
 17. A compound, according to claim 1, wherein the compound of Formula (I) is Tubulysin A.
 18. A compound, according to claim 1, wherein the polymer is a polyethylene glycol (PEG).
 19. A compound, according to claim 1, wherein the polyethylene glycol has a molecular weight of more than 30 kDa to 100 kDa. 